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IL-10-producing, ST2-expressing Foxp3(+) T cells in multiplesclerosis brain lesions
Citation for published version:Zandee, SEJ, O'Connor, RA, Mair, I, Leech, MD, Williams, A & Anderton, SM 2017, 'IL-10-producing, ST2-expressing Foxp3(+) T cells in multiple sclerosis brain lesions', Immunology and Cell Biology.https://doi.org/10.1038/icb.2017.3
Digital Object Identifier (DOI):10.1038/icb.2017.3
Link:Link to publication record in Edinburgh Research Explorer
Document Version:Peer reviewed version
Published In:Immunology and Cell Biology
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IL-10-producing, ST2-expressing Foxp3+ T cells in multiple sclerosis brain lesions 1
Running title: CD4+Foxp3+ cells in Multiple Sclerosis lesions. 2
Stephanie Elizabeth Johanna Zandee1, Richard Anthony O’Connor1, Iris Mair1, Melanie Dawn 3
Leech, Anna Williams2 and Stephen Mark Anderton1 4
Affiliations: 5
1 MRC Centre for Inflammation Research, The Edinburgh Centre for Multiple Sclerosis Research and 6
Centre for Immunity Infection and Evolution, University of Edinburgh, UK. 7
2 MRC Centre for Regenerative Medicine and The Edinburgh Centre for Multiple Sclerosis Research, 8
University of Edinburgh, UK. 9
10
Correspondence: 11
Stephen M Anderton 12
University of Edinburgh, Centre for Inflammation Research, Queen’s Medical Research Institute 13
47 Little France Crescent, Edinburgh EH16 4TJ United Kingdom 14
Phone: +44 131 242 6589 16
FAX: +44-131-242 6682 17
18
Conflict of interest statement: The authors have declared that no conflict of interest exists. 19
20
This work was supported by grants from the UK Medical Research Council, The Dutch MS Research 21
Foundation and the Scottish Chief Scientist Office. 22
23
2
Abstract 24
CD4+Foxp3+ T regulatory (Treg) cells provide a key defence against inflammatory disease, but also have 25
an ability to produce pro-inflammatory cytokines. The evidence for these two possibilities in multiple 26
sclerosis (MS) is controversial. However, this has largely been based on studies of circulating Treg cells 27
derived from peripheral blood, rather than the central nervous system. We show that Foxp3+ cells in the 28
brains of MS patients predominantly produce IL-10 and show high expression of the IL-33 receptor ST2 29
(associated with potent Treg function), indicating that Treg in the inflamed brain maintain their 30
suppressive function. 31
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Main Text 33
CD4+Foxp3+ Treg cells control immune responses in inflamed tissues as well as secondary lymphoid 34
organs1, 2. Treg cells isolated from the peripheral blood of MS patients are reported to show reduced 35
suppressive function, but not reduced frequencies3-5. Treg cells can “trans-differentiate” to a pro-36
inflammatory function, producing IFN-γ or IL-17, when placed in conducive experimental conditions6-8. 37
Peripheral blood Treg cells from MS patients were reported to display this ability, producing IFN-γ in 38
vitro under the influence of IL-129. The major drawback of such studies is that, out of necessity, only the 39
peripheral blood of MS patients can be sampled and not the central nervous system (CNS) itself. Tissue 40
inflammation can stabilize, rather than diminish Treg suppressive function. We reported that the 41
accumulation of highly activated and suppressive, IL-10-producing Treg cells within the CNS is 42
necessary for the natural resolution of experimental autoimmune encephalomyelitis (EAE), a mouse 43
model of MS10, 11. In addition, these CNS Treg cells resisted conversion to pro-inflammatory function in 44
vitro12. Here, we sought to understand the distribution of Treg in human MS lesions and to gather 45
evidence for suppressive, or pro-inflammatory roles for these cells. 46
47
Results and Discussion 48
Immunohistochemistry identified the presence of CD4+Foxp3+ T cells in post mortem brain tissue of 7/11 49
secondary progressive MS (SPMS) patients (Fig. 1a), with none found in control brain tissue. MS 50
samples that did or did not contain CD4+Foxp3+ cells could not be distinguished based on patient gender, 51
age, duration of disease, or time to post mortem processing (summarised in Supp Table 1). CD4+Foxp3+ 52
cells were distributed at similar frequencies across different white matter lesion types (9/10 active lesions, 53
3/7 chronic active borders, 3/7 chronic active centres, 9/17 chronic inactive lesions), but not in 54
remyelinating lesions (Fig. 1b). Thus, Treg presence in MS lesions appears to be associated with the 55
presence of an inflammatory infiltrate (not found in remyelinating lesions). This is consistent with our 56
previous EAE data showing that Treg numbers in the CNS decline markedly, in-line with the 57
inflammatory infiltrate, as the disease resolves10,11. Where present, the frequencies of CD4+ cells that 58
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were Foxp3+ ranged between 10-30% (Fig. 1c), which represents an enrichment over the expected 59
frequencies of these cells amongst CD4+ T cells in human peripheral blood (1-3% in healthy controls and 60
MS patients) and in cerebrospinal fluid (3-4% in MS)3, 4, 13. 61
Although the presence of CD4+IL-17+ T cells has been reported before14, no analogous analysis 62
has been made of cytokine production by Foxp3+ cells in MS lesions. Two-colour immunohistochemistry 63
identified co-expression of Foxp3 with IL-10, IL-17, IFN-γ, or GM-CSF in active and chronic lesions 64
(Fig. 2a). Approximately 50% of Foxp3+ cells stained positive for IL-10 (Fig. 2b). Lower frequencies of 65
Foxp3+ cells stained positive for pro-inflammatory cytokines. IL-10 was the dominant cytokine produced 66
by Foxp3+ cells (>60%) in active lesions and the borders of chronic active lesions (Fig. 2b). This was less 67
evident in chronic inactive lesions and in the centres of chronic active lesions, where Foxp3+ cells showed 68
no enrichment in IL-10 over other cytokines. In contrast to IL-10, TNF-α staining in Foxp3+ cells only 69
became evident in chronic inactive lesions. Frequencies of Foxp3+ cells staining for IFN-γ, IL-17, or GM-70
CSF were low in all active and chronic lesion types. We conclude that the predominant cytokine produced 71
by Foxp3+ cells within the brains of SPMS patients is IL-10. This is entirely consistent with our previous 72
observations of Treg in the CNS of mice with EAE10-12 and indicates that, in MS, Treg that infiltrate the 73
lesions are in suppressive rather than pro-inflammatory mode. 74
As CD4+Foxp3+ cells composed only a minor fraction of infiltrating cells within lesions, their 75
contribution to the overall cytokine+ cells remained modest, even for IL-10. We compared the frequencies 76
of CD4+Foxp3- or CD4+Foxp3+ cells in all lesions, with the overall levels of cytokine+ cells in those 77
lesions. CD4+Foxp3- frequencies did not correlate with any cytokine (Fig. 3a). Nor did CD4+Foxp3+ cells 78
correlate with IFN-γ, GM-CSF or IL-17. However, CD4+Foxp3+ frequencies correlated with the 79
frequencies of total IL-10+ cells and total TNF-α+ cells (Fig. 3b). 80
Elegant murine studies have shown that IL-10 signalling in Treg cells is required for their own 81
IL-10 expression and subsequent suppressive function15. Therefore it is plausible that, in addition to 82
contributing to the IL-10 pool, IL-10+ Treg cells are specifically attracted to, expanded in, or maintained 83
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in lesions with high IL-10 levels. TNF-α-blockade is a potent therapeutic option for several human 84
inflammatory diseases such as rheumatoid arthritis, Crohn’s disease and psoriasis16, 17, but not MS18. 85
Studies on how TNF-α-blockade effects the Treg populations have led to conflicting results. TNF-α 86
blockers have been reported to increase the number or function of Treg cells in RA and Crohn’s19, 20. 87
However, it has also been shown to inhibit suppressive function of Treg cells through down-regulation of 88
Foxp3 in RA patients21. Recent studies indicate TNF-α signals selectively through TNFR2 in Treg cells22, 89
23. This suggests that Treg cells might require TNF-α for their suppressive function and provides a 90
plausible explanation for the positive correlation between Foxp3+ cells and TNF-α+ cells that we see. 91
Expression of the IL-33 receptor, ST2, has been associated with potent Treg function in murine 92
models24-26. Indeed, we found ST2 to be particularly enriched in CNS Treg in EAE (Fig. 4a). IL-33 is 93
highly expressed in the CNS in both EAE and MS (Fig. 4b)27, 28. Dual immunofluorescence identified the 94
presence of Foxp3+ST2+ cells in MS brains (Fig. 4c). In particular, ~60% of Foxp3+ cells in active lesions 95
were ST2+, whilst its expression was almost absent in Foxp3+ cells in chronic lesions (Fig. 4d). High 96
expression of both IL-10 (Fig. 2b) and ST2 (Fig. 4d) by Foxp3+ cells in active lesions suggests that their 97
suppressive potency should be greatest in these lesions and that this might wane in more chronic lesions. 98
A recent study from Miron et al29 demonstrated high numbers of M2 macrophages, also particularly in 99
active lesions, of the same brain tissue studied here. This is interesting for two reasons. Firstly, IL-10 100
(perhaps originating from Treg cells) can promote the M2 phenotype, which is thought to contribute to 101
remyelination by inducing oligodendrocyte differentiation. Secondly, a study of experimental cerebral 102
malaria recently reported that IL-33 is protective by coordinating both Treg and M2 activity (the latter via 103
expansion of type-2 innate lymphoid cells which release M2-promoting cytokines)30. Whether such a 104
coordinated response is protective in CNS autoimmune inflammation, and whether there are viable 105
therapeutic approaches that can boost the numbers and/or sustain the function of these cells, should be 106
fruitful avenues for exploration. 107
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Methods 110
Human Tissue specimens 111
Post-mortem tissue from SPMS patients and control individuals who died of non-neurological causes 112
were obtained via a UK prospective donor scheme with full ethical approval and informed consent from 113
the UK Multiple Sclerosis Tissue Bank (MREC/02/2/39)(Supplementary information Table 1). Snap 114
frozen unfixed tissue blocks from 11 SPMS patients (a total of 16 blocks containing 10 active lesions, 7 115
chronic active lesions, 17 chronic inactive lesions and 12 remyelinating lesions) and 4 control blocks were 116
analysed. Lesions were classified as active, chronic active, chronic inactive and remyelinating according 117
to the International Classification of Neurological Diseases (www.icdns.org) using Luxol Fast Blue – 118
Cresyl Violet staining and Oil Red O staining. 119
Immunohistochemistry of T cell subsets 120
10 μM sections were fixed in 4% PFA (Fisher Scientific, Waltham, USA) and subsequently delipidised in 121
70% ice-cold ethanol. Antigens were retrieved using heating in acid citric buffer (Vector, Burlingame, 122
USA). Sections were incubated with anti-Foxp3 (ab10563, rabbit, Abcam, Cambridge, UK) overnight at 123
4°C. Subsequently the sections were incubated with anti-CD4 (M7310, mouse, Dako, Glostrup, Denmark) 124
for 30 minutes at room temperature. An EnVision G|2 Doublestain System, Rabbit/Mouse kit (Dako) was 125
used for detection as per manufacturer’s instructions, with exception of the use of an Vector Blue 126
Alkaline Phosphatase Substrate Kit III (Vector) to develop the signal. Sections were mounted in aqueous 127
permafluor medium (Thermo Scientific, Waltham, USA). Primary antibodies were omitted to check for 128
non-specific binding of polymers. Rabbit IgG (ab27478, Abcam) or Mouse IgG1 isotype control (X0931, 129
Dako) were used to control for non-specific binding of the primary antibodies. All IHC experiments were 130
performed in triplicate. 131
132
133
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Immunohistochemistry of cytokines 134
For double staining of Foxp3 and cytokines, combinations of antibodies against TNF-α, IFN-γ, IL-17, 135
GM-CSF or IL-10 (AF-210-NA, AF-285-NA, AF-317-NA, AF-215-NA, AF-217-NA, all goat, R&D 136
systems, Abingdon, UK) with anti-Foxp3 (rabbit, Abcam) were used. For single IL-33 staining a goat 137
anti-IL-33 antibody (AF3625, R&D systems) was used. Briefly, frozen brain sections were fixed in 4% 138
PFA (Fisher Scientific), followed by antigen retrieval as described above. Endogenous peroxidase was 139
blocked with 3% H2O2 in dH2O (Fisher Scientific), followed by blocking of biotin for 15 minutes 140
(Vector). Sections were incubated with 10% horse serum in PBS (Biosera, Boussens, France) and Fc 141
Receptor Blocking Solution was added (Human TruStain FcX Biolegend, London, UK). Primary 142
antibodies were added overnight at 4°C. Cytokines were detected with donkey anti-goat-biotin (ab6578, 143
Abcam) followed by streptavidin-alkaline phosphatase (SA-5100, Vector) and visualized with the Vector 144
Blue Alkaline Phosphatase Substrate Kit III (Vector). Slides were blocked with 10% goat serum in PBS 145
(Biosera). Anti-Foxp3 (rabbit) was detected with an anti-rabbit polymer-HRP (Dako) and developed with 146
DAB substrate (Dako). Sections were counterstained with 4',6-diamidino-2-phenylindole (DAPI) (Life 147
Technologies, Carlsbad, USA), and mounted in aqueous permafluor medium (Thermo Scientific). 148
Secondary antibodies/polymers alone, or normal goat IgG (AB-108-C, R&D Systems) and rabbit IgG 149
(Abcam) were used to control for non-specific binding. 150
151
Immunofluorescent staining of Foxp3 and ST2 152
Sections were air dried overnight, fixed in ice-cold acetone (VWR) and air dried for 30 minutes. 153
Endogenous peroxidase and biotin were blocked as described above. Sections were blocked with 10% 154
goat serum (Biosera) in PBS and incubated with rabbit anti-Foxp3 (Abcam) overnight at 4oC. Foxp3 155
antibody was detected with a goat-anti-rabbit-biotinylated antibody (BA-1000, Vector), followed by 156
incubation with a streptavidin-coupled horseradish peroxidase (SA-5004, Vector). Tyramide-Cy3 (Perkin-157
Elmer, Waltham, USA) was applied for 10 minutes to visualize the staining and ST2L FITC antibody 158
(MdBioproducts, Zürich, Switzerland) was incubated overnight at 4oC. Sections were counterstained with 159
9
DAPI (Life Technologies) and mounted in aqueous Permafluor medium (Thermo Scientific). Mouse 160
IgG1 FITC (1053002F, MdBioproducts), rabbit IgG (Abcam), or secondary antibodies/polymers alone 161
were used to control for non-specific binding. Only lesions with Foxp3+ cells were analysed. 162
163
EAE induction 164
C57BL/6 mice were bred under specific pathogen free conditions at the University of Edinburgh. All 165
experiments were approved by the University of Edinburgh Ethical Review Committee and were 166
performed in accordance with UK legislation. Female mice were used between 6-12 weeks old (n = 7). 167
EAE was induced by administration 100μg of MOG35–55 peptide (MEVGWYRSPFSRVVHLYRNGK, 168
Cambridge Research Biochemicals, Teesside, UK), emulsified in complete Freund’s adjuvant containing 169
200μg of heat-inactivated Mycobacterium tuberculosis H37Ra (Sigma-Aldrich), with a total volume of 170
100μl injected subcutaneously into the hind legs. On the same day and 48 hours later, 200ng of pertussis 171
toxin (Health Protection Agency, Dorset, UK) was given in 0.5ml of PBS intraperitoneally. Clinical signs 172
of EAE were assessed daily with the following scoring system: 0, no signs; 1, flaccid tail; 2, impaired 173
righting reflex and/or gait; 3, partial hindlimb paralysis; 4, total hindlimb paralysis; 5, hindlimb paralysis 174
with partial forelimb paralysis; 6, moribund or dead. 175
176
Isolation of CNS mononuclear cells and flow cytometry 177
Mice were sacrificed at d16 (when Treg were evident in the CNS) by CO2 asphyxiation and perfused with 178
PBS. Brains and spinal cords were removed, mechanically disrupted and digested in RPMI containing 7.5 179
mg/ml collagenase type 4 (Lorne Laboratories, Reading, UK) and 2.5 mg/ml DNAse I (Sigma-Aldrich) 180
for 30 minutes at 37°C. Mononuclear cells were isolated from the interface of a 30%:70% discontinuous 181
Percoll gradient (GE healthcare, Uppsala, Sweden) after centrifugation at 530xg for 20 minutes. Cells 182
were stained using the following antibodies: anti-CD4 brilliant violet 650 (Biolegend), anti-Foxp3 eFluor 183
450 (eBioscience, San Diego, USA), anti-ST2 FITC (MdBioscience). 184
185
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Data acquisition 186
Immunohistochemistry samples were analysed using an Olympus AX70 microscope (Olympus 187
Corporation, Tokyo, Japan). The number of cells was always quantified in the whole lesion and expressed 188
as cells per mm2 within different lesion types. The total number of nuclei was also documented. An 189
AxioScan.Z1 slide scanner (Zeiss, Cambridge, UK) was used to acquire fluorescent images and Zen Blue 190
software (Zeiss) used to process the fluorescent images. Experiments were repeated 2-3 times and 191
analysed blinded. Flow cytometric data was acquired using a Becton Dickinson (BD, Franklin Lakes, 192
USA) LSRFortessa II and analysed using FlowJo software (Tree Star version 3.2.1, Ashland, USA). 193
194
Statistical analysis 195
Where data were unevenly distributed, log transformations and statistical analysis was performed using a 196
linear mixed model. This model accounts for random effects such as having different numbers of tissue 197
blocks from each patient. In case of multiple testing, significant values were corrected with the 198
Bonferroni test. When random effects were found to be non-significant, simplified statistical tests such as 199
a Mann-Whitney-U test or a Kruskal-Wallis test were used. In case of multiple testing using a Kruskal-200
Wallis test, significant values were corrected with Dunn’s multiple comparison test. Correlations were 201
performed using Spearman rank correlation tests. Lesions were not subdivided into pathological types, 202
thereby allowing sufficient numbers for analysis. SPSS version 19 (IBM, New York, USA) statistical 203
software and Prism version 5.04 (Graphpad, La Jolla, USA) software were used to perform the 204
calculations. Data are presented as mean ± SEM. Significant differences are denoted as * p<0.05, ** 205
p<0.01 and *** p<0.001. 206
207
Acknowledgements: We thank the UK Multiple Sclerosis Tissue Bank for providing human brain tissue, 208
F. Roncaroli (Imperial College London) for neuropathological diagnosis, R. Nicholas (Imperial College 209
London) for providing clinical histories, Mr. D.M. Mole for collaboration in developing several 210
immunohistochemistry techniques, Ms. A. Boyd for technical assistance, Dr. M. Chase-Topping for 211
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statistical advice and staff of Flow Cytometry and Histology/Imaging Facilities. This work was supported 212
by the Scottish Chief Scientist Office (ETM/163), the Dutch MS Foundation (r12-1MS) and the UK 213
Medical Research Council (G0801924). 214
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Conflicts of interest: The authors have no conflicting interests to declare. 216
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Supplementary information is available at the Immunology and Cell Biology website. 218
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References 220
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Figure legends 295
Figure 1. CD4+Foxp3+ T cell are enriched within MS lesions. 296
(a) Representative images from different MS cases (A = active, CA = chronic active and CI = chronic 297
inactive lesions) of immunohistochemistry for CD4 (blue) and Foxp3 (brown). No staining was observed 298
using isotype controls or secondary antibodies alone. Scale bars 20 µm. Accompanying images show 299
lesions (LFB = Luxol Fast Blue – Cresyl Violet). Dotted line represents lesion border. Black boxes 300
delineate the areas CD4+Foxp3- and CD4+Foxp3+ cells were pictured. Scale bars 200 µm. (b) Densities of 301
CD4+Foxp3- and CD4+Foxp3+ cells in the indicated SPMS lesion types. (c) Frequencies of CD4+ cells that 302
were Foxp3+ in the indicated SPMS lesion types. Graphs show means ± SEM. Kruskal-Wallis tests with 303
Dunn’s multiple comparison correction were used. * p<0.05, ** p<0.01. 10 active lesions, 7 chronic 304
active lesions, 17 chronic inactive lesions and 12 remyelinating lesions were studied. 305
306
Figure 2. Foxp3+ cells predominantly produce IL-10 in MS lesions. 307
(a) Representative images of immunohistochemistry for individual cytokines (blue) and Foxp3 (brown). 308
No staining was observed using isotype controls or secondary antibodies alone. Scale bars 20 µm. (b) 309
Frequencies of Foxp3+ cells co-staining for individual cytokines in the indicated SPMS lesion types. 310
Graphs show means ± SEM. A Kruskal-Wallis test with Dunn’s multiple comparison correction was used. 311
** p<0.01. 5 active lesions, 3 chronic active lesions and 8 chronic inactive lesions were studied. 312
313
Figure 3. Frequencies of CD4+Foxp3+ cells correlate with IL-10 and TNF-α levels in MS lesions 314
Relationships between the frequencies of CD4+Foxp3- cells (a), or CD4+Foxp3+ cells (b), and the 315
frequencies of all cells staining for the indicated cytokine. Non-parametric 2-sided Spearman correlations 316
were used. Lesions were not segregated based on pathological type. 10 active lesions, 7 chronic active 317
lesions and 17 chronic inactive lesions were studied. 318
319
Figure 4. Foxp3+ST2+ Treg are present in MS lesions. 320
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(a) Representative flow cytometry plots (gated on CD4+ cells) and summary data showing the expression 321
of ST2 in CD4+Foxp3+ cells in spleen, lymph nodes (LN) and CNS isolated from mice 16 days after 322
induction of EAE. A one-way ANOVA with Bonferroni’s post test was used. Graphs show means ± 323
SEM. 7 mice were studied. (b) Representative immunohistochemistry image of IL-33 (brown) and 324
haematoxylin (blue) and summary data showing percentage of IL-33+ cells in the indicated human SPMS 325
lesions. No staining was observed using isotype controls. Scale bars 20 µm. (c) Representative 326
immunofluorescent staining for DAPI (blue), ST2 (green) and Foxp3 (red) in an active lesion. Arrows 327
delineate ST2+Foxp3+ cells (insets). No staining was observed using isotype controls. Scale bars 40 µm. 328
(d) Frequencies of Foxp3+ cells that stained for ST2 in the indicated SPMS lesions. 5 active lesions, 2 329
chronic active lesions and 6 chronic inactive lesions were studied. A Kruskal-Wallis test with Dunn’s 330
multiple comparison correction was used. * p<0.05. 331
332